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First published online 11 October 2006
doi: 10.1242/dev.02644
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1 Keratinocyte Laboratory, Cancer Research UK London Research Institute, 44
Lincoln's Inn Fields, London WC2A 3PX, UK.
2 Department of Immunology, Tokai University School of Medicine, 143
Shimokasuya, Isehara, Kanagawa 259-1193, Japan.
Author for correspondence (e-mail:
fiona.watt{at}cancer.org.uk)
Accepted 14 September 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: ß-catenin, Notch, Jagged 1, Epidermis, Mouse
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Epidermal stem cells self-renew and generate transit amplifying cells that
exit the cell cycle after a few rounds of division and undergo terminal
differentiation along the multiple lineages that constitute the hair follicle,
interfollicular epidermis (IFE) and sebaceous gland. Several evolutionarily
conserved pathways that regulate embryonic skin development are important in
postnatal life, including the Wnt, hedgehog, Notch and Bmp pathways
(Fuchs et al., 2004). It is
now clear that varying the strength, timing and duration of an individual
signal exerts different effects on the epidermal stem cell compartment
(Lowry et al., 2005;
Silva-Vargas et al., 2005).
However, little is known about how multiple pathways intersect at the level of
individual cells. In this report we have investigated the interaction between
the Wnt and Notch signalling pathways in the skin.
A key component of the canonical Wnt pathway is ß-catenin
(Reya and Clevers, 2005). In
the absence of Wnt, ß-catenin is degraded in the cytoplasm. When Wnt
binds to its cell surface receptors, the ß-catenin destruction complex is
inactivated, allowing ß-catenin to enter the nucleus, where it binds to
transcription factors of the Lef/Tcf family and activates transcription.
Inhibition of Wnt signalling prevents hair follicle formation, whereas
activation of ß-catenin in postnatal epidermis induces de novo follicles
(Alonso and Fuchs, 2003;
Lo Celso et al., 2004).
Depending on the level of ß-catenin activation and cellular context,
ß-catenin can expand the epidermal stem cell compartment
(Zhu and Watt, 1999) or
trigger stem cells to become transit amplifying cells
(Lowry et al., 2005) and form
ectopic hair follicles in adult epidermis
(Alonso and Fuchs, 2003;
Silva-Vargas et al.,
2005).
An essential aspect of Notch signalling is a proteolytic cleavage event
mediated by 
-secretase activity
(Artavanis-Tsakonas et al.,
1999). On interaction of the Notch receptor with its ligands
-secretase liberates the Notch intracellular domain (NICD).
NICD translocates to the nucleus and heterodimerises with RBP-J
(CSL, CBF1; Rbpsuh - Mouse Genome Informatics), thereby activating
target gene transcription. In contrast to Wnt, Notch signalling is not
essential for embryonic specification of hair follicles; however, Notch is
required for subsequent hair follicle maintenance
(Lin et al., 2000;
Pan et al., 2004;
Uyttendaele et al., 2004;
Vauclair et al., 2005).
Whereas ß-catenin activation is linked to skin tumour formation
(Alonso and Fuchs, 2003),
Notch1 acts as a tumour suppressor
(Nicolas et al., 2003) and
promotes differentiation of keratinocytes in vitro
(Lowell et al., 2000;
Rangarajan et al., 2001).
Based on the gain- and loss-of-function studies of Wnt and Notch in mouse
epidermis, it is likely that the pathways interact in controlling hair
follicle differentiation. Several potential mechanisms would place Notch
upstream of Wnt. Notch1 activation is reported to suppress epidermal
expression of Wnt4 (Devgan et al.,
2005). In Drosophila, the region of Notch C-terminal to
the cdc10/ankyrin repeats associates with Armadillo/ß-catenin and
negatively regulates ß-catenin transcriptional activity
(Hayward et al., 2005). To
elucidate potential Wnt/Notch crosstalk within the epidermis, we examined
where Notch is active, the impact of Notch loss of function on
ß-catenin-induced hair follicle formation, and the consequences of
simultaneously activating both pathways in the same cells.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) were
crossed with K5Cre mice (gift of Jose Jorcano)
(Ramirez et al., 2004),
K14CreER mice (gift of Barry Stripp) (Hong
et al., 2004) and K14
N ß-cateninER mice (D2 line)
(Lo Celso et al., 2004). TNR
mice, which contain a transgene that acts as a GFP reporter of Notch activity
(Duncan et al., 2005), were the
gift of N. Gaiano. K14NLef1 mice were also examined
(Niemann et al., 2002).
The NICDOPER transgene consists of amino acids 1751-2290
of the mouse Notch1 intracellular domain (NICDOP) with an
N-terminal FLAG sequence and a modified oestrogen receptor (ER) fused in frame
at the C terminus (gifts of U. Just and P. Chambon)
(Schroeder and Just, 2000).
The NICDOPER construct was subcloned into the BamHI
restriction site of the Keratin 14 (K14) promoter cassette provided by E.
Fuchs (Vasioukhin et al.,
1999) and injected into the pronucleus of (CBAxC57bl) F1
embryos. Potential founder lines were screened by PCR (forward: rabbit
ß-globin intron 5'-TACTCTGAGTCCAAACCGGGC-3' and reverse:
mouse Notch1 5'-CACTCGTTCTGATTGTCGTC-3') and confirmed by Southern
blotting. Founder lines 5199.15A,B+C (CA3, low copy number) and 5199.21A,B+C
(CA4, high copy number) were established; both lines had a similar phenotype,
the severity correlating with copy number.
Line 5199.21A,B+C was used to characterise the
K14NICDOPER phenotype. We examined 34 4OHT-treated
transgenic mice, 23 acetone-treated transgenic mice and 18 4OHT-treated
wild-type mice. The animals were analysed between 6 and 13 weeks of age.
To activate transgenes that contained an ER domain, mice were treated
topically with 1-3 mg 4OHT (Sigma) as described
(Lo Celso et al., 2004). Some
mice were treated with 1 mg N-S-phenyl-glycine-t-butyl ester (DAPT, Sigma),
dissolved in 1% DMSO/acetone. When mice were treated with DAPT and 4OHT, DAPT
was applied 30 minutes before 4OHT.
Histology and immunohistochemistry
Whole mounts, frozen sections and paraffin-embedded sections were prepared
and immunolabelled as described previously
(Braun et al., 2003;
Lo Celso et al., 2004).
Antibodies to the following proteins were used: CCAAT displacement protein
(CDP, a gift of M. Busslinger, IMP, Vienna, Austria), Ki67 (Vector
Laboratories), trichohyalin (AE15, a gift of T. T. Sun, NYU Medical Center,
New York, USA), Jag1 (Santa Cruz Biotechnologies), keratin 14 (MK14, Covance),
keratin 10 (MK10, Covance), ß1 integrin (MB1.2, a gift of B. Chan,
University of Western Ontario, London, Canada), oestrogen receptor (HC-20,
Santa Cruz Biotechnologies), green fluorescent protein (GFP, Molecular Probes
or Abcam), inner root sheath keratins (AE13; a gift of T. T. Sun), cleaved
Notch1 (Val 1744, Cell Signaling Technologies or AB8925, Abcam) and
ß-catenin (Santa Cruz Biotechnologies). Haematoxylin or DAPI was used as
nuclear counterstain.
In whole mounts of tail skin, NICD was detected using the TSA Plus Amplification Kit (Perkin Elmer). Tail skin was incubated with primary antibody overnight, followed by an overnight incubation with an HRP-conjugated anti-rabbit secondary antibody (Amersham), reacted with fluorescein-conjugated tyramide and mounted with Mowiol.
Luciferase assays
Spontaneously immortalised keratinocytes from wild type and K14N
ß-cateninER mice were grown on a feeder layer of J2-3T3 cells
(Lo Celso et al., 2004). To
measure Notch and ß-catenin dependent transcription,
8x104 or 2x105 cells were plated per 35 mm
well in KSFM. Cells were transfected using Lipofectamine 2000 (Invitrogen)
with Hes1 luciferase, pCDNA3 mNotch1ICD, pCDNA3 hNotch1 or TOPFlash
luciferase. FOPFlash luciferase, pRL and pCI empty vectors served as controls.
Post-transfection, cells were cultured in KSFM with or without 200 nmol/l 4OHT
and/or 1 µmol/l DAPT. Forty-eight or 72 hours after transfection,
luciferase levels were measured as previously described
(Silva-Vargas et al., 2005).
Statistical significance was determined with Student's t-test using
QuickCalcs software (GraphPad Software).
RT-PCR
Spontaneously immortalised mouse keratinocytes were plated for 48 hours in
KSFM (Gibco) and then treated with cycloheximide (25 µmol/l) for 30
minutes. Cells were incubated for a further 4 hours in medium containing
cycloheximide and 200 nmol/l 4OHT (Sigma) or acetone. Total RNA was isolated
from cells using TriReagent (Helena Bioscience), and subjected to reverse
transcription using Superscript reverse transcriptase (Invitrogen). cDNAs were
amplified by PCR using the following oligonucleotide pair as Jag1
primers: 5'-TCCAGGTCTTACCACCGAAC-3' and
5'-GGACGCCTCTGAACTCTGAC-3'. Mouse ß-actin primers were
described previously (Giangreco et al.,
2004).
Jag1 promoter analysis
We used TESS software to analyse the promoter sequence of Jag1.
This software is freely available online at:
http://www.cbil.upenn.edu/cgibin/tess/tess.
For ChIP assays, spontaneously immortalised mouse keratinocytes were plated without feeders for 48 hours in complete medium and then treated with 200 nmol/l 4OHT for 4 hours. 1x106 cells were used per immunoprecipitation and processed using a ChIP assay kit (Upstate) according to the manufacturer's instructions. Immunoprecipitations were performed using a rabbit antibody to ß-catenin (Santa Cruz Biotechnologies) and an isotype-matched control antibody with an overnight incubation at 4°C. Immunoprecipitated DNA was amplified using the following PCR primer pairs specific for the putative Tcf/Lef binding sites or irrelevant sequence in the mouse Jag1 promoter: 4827-4334: 5'-GCCAAACCAACGCTTAACAT-3'; 5'-CATAGCCAGAGGCAGACACA-3'; 3580-3157: 5'-TCCATCCCCCAATTAAGACA-3'; 5'-TGATGCCTCAATTCCCTTTC-3'; 2958-2553: 5'-TTCAGGGGTGATCAAGGAAG-3'; 5'-GAGCCAGCCACCTGAGTTAC-3'; 2618-2139: 5'-TTTCACGAAGCCCAGATTGT-3'; 5'-ATTTTCCAGTGTGCCCAGTC-3'; 1778-1359: 5'-TAGAAGGGTTGAGGCGCTAA-3'; 5'-CTTTCCCGGAGTCAGACTTG-3'; 831-370: 5'-CCTTCCAGGTTCCTTTCTCC-3'; 5'-CCTCTCGGCTTTCTTTCCTT-3'.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) to
determine where Notch was active in adult epidermis
(Fig. 1A-C). GFP was detected
in the pre-cortex of growing (anagen) hair follicles, the cuticle of the inner
root sheath, the outer root sheath and the dermal papilla
(Fig. 1A). The pre-cortex is
the zone at the base of the follicle in which selection of most of the hair
follicle lineages takes place (Legue and
Nicolas, 2005). As previously reported
(Pan et al., 2004;
Vauclair et al., 2005), the
pre-cortex was positively labelled with an antibody to NICD
(Fig. 1D,E), confirming that it
is a region of Notch signalling activity. The same region is known to express
Lef1 and to be a site of ß-catenin reporter activity
(DasGupta and Fuchs, 1999).
Notch-GFP reporter activity, and positive staining for NICD, was
also detected in small clusters of cells of the basal layer of interfollicular
tail epidermis and throughout the suprabasal layers
(Lowell et al., 2000)
(Fig. 1B,C; data not
shown).
The Notch ligand, Delta1, is expressed during embryonic hair follicle
development but not in postnatal skin
(Favier et al., 2000;
Powell et al., 1998). In adult
skin jagged 1 was expressed in the pre-cortex and matrix (the proliferative
zone below the pre-cortex) of growing hair follicles, in a stripe of cells
along the outer root sheath, and in clusters of cells in the IFE
(Fig. 1A,F,G). Therefore, the
locations of jagged 1 expression corresponded to the locations of Notch
activity in postnatal epidermis. Jagged 1 expression and Notch activity were
elevated in regions of the epidermis where cells were undergoing commitment to
terminal differentiation and thus appear to be associated with transit
amplifying cells rather than stem cells
(Legue and Nicolas, 2005;
Lowell et al., 2000).
Consequences of inhibiting or activating the Notch pathway
To examine the role of Jag1 in adult mouse skin, we crossed
Jag1flox/flox mice (Brooker et
al., 2006) with K5Cre mice
(Fig. 1H,P). As expected, Jag1
protein was not detected in K5Cre Jag1flox/flox epidermis
(Fig. 1H). In addition, hair
follicle staining with an antibody to NICD (activated Notch) was
reduced on deletion of Jag1 (Fig.
1I,J). The K5Cre Jag1flox/flox mice appeared normal at
birth but started to lose their hair and whiskers at 5 weeks and were
completely bald at 7.5 weeks. Subsequent hair growth was sparse and the hairs
were short, resembling the Notch1 deletion phenotype
(Vauclair et al., 2005)
(Fig. 1P).
To activate the Notch pathway in keratinocytes, we used a truncated form
(amino acids 1751-2290) of the intracellular domain of mNotch1
(NICDOP) coupled to a modified form of the human oestrogen
receptor (ER). This activatable form of Notch lacks the C-terminal region (OPA
and PEST domains) but contains the RAM/ankyrin repeats that mediate
interaction with RBP-J and transcription of Notch target genes
(Schroeder and Just, 2000). In
vitro, mouse keratinocytes transiently transfected with the
NICDOPER construct activated a Notch-responsive Hes1
luciferase reporter only in the presence of 4-hydroxytamoxifen (4OHT)
(Fig. 1K).
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OPER under the control
of the K14 promoter (Fig.
1M-O,R). When sections of epidermis from transgene-negative mice
were stained with an antibody to the ER, no staining was detected
(Fig. 1L). In
K14NICDOPER transgenic mice treated with acetone, the ER
epitope was detected in the cytoplasm of cells in the regions of known K14
promoter activity: the basal layer of the IFE, the outer root sheath of the
hair follicle and the periphery of the sebaceous glands
(Fig. 1M; data not shown). In
addition there was weak suprabasal staining in some regions of the IFE,
possibly due to the long half-life of the protein in the absence of 4OHT
(Fig. 1M). In transgenic mice
treated with 4OHT, ER expression was, as predicted, predominantly nuclear in
transgenepositive cells in the IFE, sebaceous gland and hair follicle, and no
suprabasal nuclear staining was observed
(Fig. 1N,O; data not
shown).
Whereas Jag1 deletion resulted in hair loss
(Fig. 1P), Notch activation
resulted in clumping of hair follicles, which was readily observed
macroscopically (Fig. 1Q,R). At
6.5-10 weeks, the hair follicles of wild-type mice were in the resting
(telogen) phase of the hair cycle and 4OHT treatment did not stimulate hair
re-growth (Fig. 1Q). After 14
days of 4OHT treatment, K14NICDOPER transgenic mice did not
enter anagen, but their follicles were abnormally clustered, and the hairs
were not uniformly oriented (Fig.
1R). The patchy phenotype was not due to low Notch activity,
because it was seen in both homozygous and heterozygous transgenic mice (data
not shown), and did not reflect inefficient induction of the transgene, as
evidenced by the nuclear localisation of NICDOPER
(Fig. 1N,O). The fact that the
mice failed to exhibit uniform hair re-growth in response to 4OHT treatment
demonstrates that activation of Notch, unlike ß-catenin
(Lo Celso et al., 2004), is
not sufficient to induce anagen.
Notch signalling is required for differentiation of hair follicle lineages
We analysed the histology of Jag1flox/flox, K5Cre
Jag1flox/flox and K14NICDOPER back skin and
performed immunolabelling with antibodies to differentiation markers. As
controls, K14NICDOPER mice were treated with acetone, and
Jag1flox/flox and wild-type mice were either untreated or treated
with acetone or 4OHT. All controls were indistinguishable from untreated
wild-type skin (Fig. 2A,B; data
not shown).
K5Cre Jag1flox/flox back skin was examined at 5 weeks
(Fig. 2C). In contrast to
Jag1flox/flox follicles, which were in anagen
(Fig. 2A), the K5Cre
Jag1flox/flox follicles were in the resting (telogen) state. Seven
and a half-week-old mice that were bald
(Fig. 1P) had normal sebaceous
glands, but developed epidermal cysts (Fig.
2D). The hair follicles failed to enter anagen and were thinner
than normal (Fig. 2E).
Conversely, the IFE was thicker in Jag1-null than wild-type epidermis
(Fig. 2B,E). At 10 weeks, when
control epidermis was once more in telogen
(Fig. 2B), some hair regrowth
was observed in K5Cre Jag1flox/flox mice, but the hairs were sparse
and short (data not shown). This correlated with the presence of epidermal
cysts and abnormal hair follicles (Fig.
2E), and resembled the phenotype of mice with epidermal deletion
of -secretase, Notch1 or both Notch1 and
Notch2 (Pan et al.,
2004; Vauclair et al.,
2005).
We next performed histological analysis of the skin of 10- to 13-week-old
4OHT-treated K14NICDOPER mice. This confirmed that the hair
follicles were unevenly spaced (Fig.
1R), with multiple follicles often in close proximity
(Fig. 2F). The patchy
distribution of the follicles might be caused by changes in the
interfollicular epidermal surface area between follicles, although this
remains to be investigated. Similar to 10-week-old Jag1flox/flox
mice, which had a wild-type phenotype, the follicles in 4OHT-treated
transgenic mice were mainly in telogen
(Fig. 2B,F). This supports the
conclusion that Notch is not sufficient to induce hair follicle growth.
In treated K14NICDOPER transgenic mice that were of an
age (13 weeks) at which hairs had re-entered anagen, enlarged hair follicle
bulbs and sebaceous glands were found (Fig.
2G,H). In many cases the enlarged bulbs appeared to have detached
from the rest of the follicle, forming cysts resembling massive hair bulbs
with aberrant hair shafts (Fig.
2H).
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), was
expressed in the suprabasal layers of normal IFE
(Fig. 3A). We observed
increased numbers of K10-positive layers in the thickened K5Cre
Jag1flox/flox IFE from back and tail skin compared with controls
(Fig. 3B; data not shown).
Jag1-null cysts were filled with cornified material
(Fig. 2E) and were K10-positive
(Fig. 3D). Although the IFE of
4OHT-treated K14NICDOPER mice was also thicker than
wild-type epidermis (Fig.
2F-H), expression of K10 was lost in large areas and there was a
corresponding increase in the number of K14-positive cell layers
(Fig. 3C; data not shown). The
loss of K10 in K14NICDOPER epidermis did not reflect a
failure of IFE differentiation, because the cornified layers appeared normal
(Fig. 3C). Instead, loss of K10
correlated with epidermal hyperproliferation, as judged by an increased number
of Ki67-positive cells and expression of keratin 17 (data not shown).
|
).
Expression of CDP (Fig.
3H-K,Q,U) and trichohyalin
(Fig. 3L-O) was lost in K5Cre
Jag1flox/flox epidermis, and whole-mount labelling of tail
epidermis (Braun et al., 2003)
confirmed that the hair follicles were thinner than controls
(Fig. 3P,T). Jag1 null
cysts also lacked expression of CDP, trichohyalin and inner root sheath
keratins (data not shown). By contrast, the enlarged hair bulbs in
K14NICDOPER epidermis were CDP-positive, and expressed
trichohyalin and the inner root sheath keratins detected with antibody AE13
(Lo Celso et al., 2004)
(Fig. 3E-G).
Whereas Notch activation in 4OHT-treated K14NICDOPER
epidermis resulted in enlarged sebaceous glands
(Fig. 2F), Jag1
deletion did not affect the size of the sebaceous glands. However, when
visualised by Nile Red staining of tail epidermal whole mounts,
Jag1-null sebaceous glands were somewhat irregular in shape
(Fig. 3R,V).
To examine whether there was an ongoing requirement for Jag1 in adult epidermis, we crossed Jag1flox/flox mice with K14CreER mice and deleted Jag1 by topical application of 4OHT (Fig. 3S,W). Mice were treated with 4OHT or acetone for 3 weeks, starting when they were 8 weeks old. At 11 weeks, 54% of hair follicles in control, acetone-treated tail epidermis were entering anagen, as evaluated by strong CDP expression and the morphology of follicles in epidermal whole mounts (data not shown; Fig. 3S). By contrast, following deletion of Jag1, the number of anagen follicles in 11-week-old mice was reduced to 6.5% (Fig. 3W). We conclude that there is an ongoing requirement for Jag1 during the postnatal hair cycles.
The phenotypes of the K5Cre Jag1flox/flox and the
K14NICDOPER mice suggest that Notch signalling is required
for, and promotes, differentiation of the mature hair follicle lineages,
consistent with the observation that -secretase is required to maintain
the inner root sheath cell fates (Pan et
al., 2004). The similarities between epidermis lacking
Notch1 (Vauclair et al.,
2005) and epidermis lacking Jag1
(Fig. 2) identify jagged 1 as
the key ligand for Notch signalling in postnatal epidermis.
Notch activity is required downstream of ß-catenin for ectopic hair follicle formation
Unlike ß-catenin activation, Notch activation did not promote anagen
or induce ectopic follicles, and this would tend to place Notch downstream of
ß-catenin. To investigate how the two pathways interacted, we examined
microarrays of genes induced in response to ß-catenin activation in
K14N ß-cateninER mice
(Silva-Vargas et al., 2005).
Jag1 and the downstream Notch targets, Hes and Hey family members
(Iso et al., 2003), were
upregulated upon ectopic ß-catenin activation
(Table 1). The array results
were confirmed by RNA in situ hybridisation (data not shown).
|
N ß-cateninER
mice with 4OHT in combination with the -secretase inhibitor,
N-S-phenyl-glycine-t-butyl ester (DAPT)
(Cheng et al., 2003). Second,
we generated triple transgenic mice by crossing Jag1flox/flox mice,
K14CreER mice (Hong et al.,
2004) and K14N ß-cateninER mice
(Lo Celso et al., 2004). We confirmed that DAPT inhibited Notch activation in vivo by performing western blots on protein lysates of mouse skin treated topically for 1 or 2 hours with DAPT or the vehicle control (Fig. 4A). At each time point the level of NICD was reduced in treated, compared with untreated, skin. The effect was, however, transitory, as 4 hours after DAPT application the level of NICD was indistinguishable from untreated control skin (data not shown).
|
N ß-cateninER
keratinocytes treated with 4OHT (Fig.
4C).
K14N ß-cateninER mice were treated with acetone, 4OHT or 4OHT
plus DAPT for 15 days. In agreement with previous observations
(Lo Celso et al., 2004), hair
follicles in 4OHT-treated K14N ß-cateninER mice entered anagen and
de novo follicles formed from preexisting follicles, sebaceous glands and the
IFE (Fig. 4D,F), while hair
follicles in wild-type or acetone-treated transgenic animals (data not shown)
remained in telogen. In K14N ß-cateninER epidermis treated with
4OHT and DAPT, the onset of anagen of existing hair follicles and de novo hair
follicle formation were severely delayed
(Fig. 4E,G). Moreover, ectopic
expression of CDP was reduced in K14N ß-cateninER mice treated
with DAPT (Fig. 4F,G).
In the Jag1flox/floxxK14CreERxK14N
ß-cateninER triple transgenics, application of 4OHT deleted Jag1
and activated ß-catenin in cells in which the K14 promoter was active.
Immunostaining of back skin sections with antibodies to jagged 1 and
ß-catenin confirmed that activation of ß-catenin and Jag1
deletion occurred in the same cellular compartments
(Fig. 5D-I). Nuclear
ß-catenin was detected in the IFE of triple transgenics
(Fig. 5I) and K14N
ß-cateninER single transgenics (Fig.
5G), but not in the Jag1flox/flox controls
(Fig. 5E).
|
The effects of deleting Jag1 while simultaneously activating
ß-catenin were examined further by preparing tail epidermal whole mounts
from Jag1flox/flox (wild-type phenotype), K14N
ß-cateninER and triple transgenics treated with 4OHT for 21 days
(Fig. 5J-U). As in back skin
(Fig. 5C),
ß-catenin-induced ectopic hair follicle formation was completely blocked
by removal of Jag1 (Fig.
5J,N,R). Nile Red staining showed that the reduction in
differentiated sebocytes characteristic of K14N ß-cateninER
epidermis (Lo Celso et al.,
2004) was prevented in triple transgenics, although sebaceous
gland morphology was slightly disturbed relative to Jag1flox/flox
epidermis (Fig. 5K,O,S).
Ectopic expression of CDP was inhibited in the triple transgenics
(Fig. 5P,T). In triple
transgenics, consistent with the failure of the follicles to enter anagen, CDP
expression was also absent from the bulb
(Fig. 5L,T).
Pharmacological inhibition of hedgehog signalling blocks ectopic hair
follicle formation in K14N ß-cateninER epidermis by inhibiting
ß-catenin-induced proliferation
(Silva-Vargas et al., 2005).
By contrast, inhibition of Notch signalling in the triple transgenics did not
prevent ß-catenin-induced proliferation in the sebaceous glands and IFE,
as evaluated by Ki67 staining (Fig.
5M,Q,U). We observed clusters of highly proliferative cells in the
IFE and in the sebaceous glands in K14N ß-cateninER mice and
triple transgenics. There was a reduction in proliferation in the hair
follicle bulb, but that correlated with the failure of the triple transgenic
follicles to enter anagen (Panteleyev et
al., 2001) (Fig.
5U). We conclude that while Notch, like hedgehog, is required
downstream of ß-catenin for ectopic hair follicle formation, its role
differs from hedgehog in that it is not primarily a proliferative
stimulus.
Co-operation between Notch and ß-catenin in promoting ectopic hair follicle formation
If Notch acts primarily to promote differentiation of the hair follicle
lineages downstream of ß-catenin, simultaneous activation of
ß-catenin and Notch should give rise to ectopic follicles that are more
mature than those resulting from activation of ß-catenin alone. To
examine this, we crossed K14N ß-cateninER and
K14NICDOPER mice to generate double transgenics. In response
to 4OHT, hair follicles entered anagen in both the double and the K14N
ß-cateninER single transgenics, while hair follicles in 4OHT-treated
wild-type skin remained in telogen (Fig.
6A-C).
We determined the number of patches of CDP expression per existing follicle
as a means of quantitating ectopic hair follicle formation, as described
previously (Fig. 6D-F)
(Silva-Vargas et al., 2005).
There were more follicles in double transgenics than in single K14N
ß-cateninER transgenics at 8 days of 4OHT treatment
(P<0.003); however, by 15 days there was no significant difference
(P<0.5; Fig. 6J).
Thus, although there were initially more ectopic follicles in the double
transgenics, there was no significant difference at the later time point. One
possible explanation for this result is that Notch activation lowers the
threshold of ß-catenin activation required to initiate, but not to
maintain, an ectopic follicle.
At all time points the ectopic follicles were longer and there were more
Ki67-positive cells in double than in single transgenic epidermis
(Fig. 6G-I). As previously
reported (Lo Celso et al.,
2004), ß-catenin-induced follicles lacked the inner root
sheath marker, trichohyalin (Fig.
6K). However, ectopic follicles in the double transgenics were
frequently trichohyalin positive and thus more mature
(Fig. 6L). We conclude that
Notch activation promotes hair follicle differentiation in the presence of
both endogenous (Fig. 3) and
ectopic ß-catenin (Fig. 6)
signalling.
To confirm that ß-catenin activation stimulates Notch activity, we
crossed K14N ß-cateninER and Notch reporter mice (TNR) and
examined expression of GFP as a readout of Notch activity. As controls we also
examined GFP expression in TNR single transgenics and in 4OHT-treated,
K14NICDOPER/TNR double transgenics. As shown in
Fig. 1A-C, GFP expression was
detected in TNR single transgenic mice in a subset of interfollicular basal
and suprabasal cells and regions of the hair shaft
(Fig. 6M,O). In 4OHT-treated
K14NICDOPERxTNR double transgenic mice GFP was
expressed in all sites where the K14 promoter is active: throughout the basal
layer of the IFE, along the hair follicle outer root sheath and at the
periphery of the sebaceous glands (Fig.
1M-O and Fig.
6N,P). In K14N ß-cateninERxTNR double transgenic
mice, GFP expression was upregulated in the existing hair follicles and in
ectopic follicles, but not in phenotypically normal IFE
(Fig. 6Q). This confirms that
Notch signalling is activated in response to ß-catenin activation in hair
follicles.
Jag1 is a ß-catenin target gene
We next examined the mechanism by which the Notch pathway is upregulated by
ß-catenin activation. We found that jagged 1 protein
(Fig. 7A,B) and mRNA (data not
shown) were upregulated when K14N ß-cateninER epidermis was
treated with 4OHT. When 4OHT treatment was stopped, ectopic follicles
regressed (Silva-Vargas et al.,
2005) and jagged 1 expression was downregulated, becoming confined
to the bulb of preexisting follicles (Fig.
7C). When 4OHT treatment was restarted, jagged 1 expression was
again increased (Fig. 7D,E).
This shows a very tight coordination of ß-catenin activation and jagged 1
expression. Although Jag1 is positively regulated by Notch
(Ross and Kadesch, 2004), in
K14N ß-cateninER epidermis treated simultaneously with 4OHT and
DAPT there was increased jagged 1 expression, even though ectopic hair
follicle formation and Notch activation were inhibited
(Fig. 7F,G).
Further evidence that ß-catenin might directly regulate Jag1
came from examining the skin of K14NLef1 mice, in which inhibition of
ß-catenin signalling results in conversion of hair follicles into cysts
of IFE with associated sebocytes (Niemann
et al., 2002). Jagged 1 expression was undetectable in the
epidermis of 3-monthold K14NLef1 transgenics, an age at which the
follicles had converted into epidermal cysts
(Fig. 7H-J). Moreover, jagged 1
expression was already lost in tail epidermis of K14NLef1 mice by 8
weeks of age (Fig. 7L,M).
When keratinocytes were transiently transfected with the full-length Notch1
intracellular domain (mNotch1ICD) and a luciferase reporter of
Notch activity (Hes1 luciferase), activation of ß-catenin by 4OHT in
K14N ß-cateninER cells resulted in a significant increase
(P<0.01) in luciferase activity compared with 4OHT-treated
wild-type cells (Fig. 7K). As
this effect was more pronounced at 72 hours post-transfection than at 24
hours, we speculate that the increase in luciferase activity is dependent on
endogenous jagged 1 production.
Analysis of the sequence of the mouse Jag1 promoter revealed five
putative Tcf/Lef-binding sites with the same consensus sequence
(Fig. 7N). Comparison with the
human and rat Jag1 promoters revealed the presence of, respectively,
three and six putative binding sites with the same consensus sequence
(CCTTTG). Their respective locations on the promoter suggest functional
conservation between species. Our results are in good agreement with those of
Katoh and Katoh (Katoh and Katoh,
2006), who have reported conservation of Tcf/Lef-binding sites
within the promoter of mammalian Jag1 orthologues.
|
N ß-cateninER, but not
wild-type, keratinocytes treated with 4OHT in the presence of cycloheximide,
providing evidence that Jag1 is upregulated by ß-catenin at the
transcriptional level (Fig.
7O). Jag1 was confirmed as a ß-catenin target gene
by performing a ChIP assay using an anti-ß-catenin antibody and wild-type
and K14N ß-cateninER mouse keratinocytes
(Fig. 7P). ChIP PCR bands were
observed using different pairs of oligonucleotides that covered the five
putative Tcf/Lef-binding sites identified in
Fig. 7N. All the binding sites
were also occupied by ß-catenin in wild-type cells, suggesting that the
activation of Jag1 by ß-catenin occurs in response to endogenous
Wnt signalling and not solely as a consequence of ß-catenin
overexpression. As Dll1 is transcriptionally regulated by ß-catenin
during embryonic development (Galceran et
al., 2004; Hofmann et al.,
2004), we conclude that ß-catenin regulates expression of
both the embryonic and postnatal epidermal ligands of Notch. |
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
) and
intestine (Fre et al., 2005),
the Wnt and Notch signalling pathways are closely linked. Both pathways must
be active in order to maintain postnatal hair growth, and if either is blocked
hair follicles convert into cysts of IFE
(Fig. 2)
(Huelsken et al., 2001;
Merrill et al., 2001;
Niemann et al., 2002;
Vauclair et al., 2005;
Yamamoto et al., 2003). When
both pathways are activated simultaneously, there are initially more ectopic
follicles than when only ß-catenin is active. However, the increase is
only temporary, suggesting that Notch activation may lower the threshold of
ß-catenin activity required to initiate, but not to maintain, an ectopic
follicle (Lowry et al., 2005;
Silva-Vargas et al., 2005).
Overall, our data suggest that the effect of Notch activation is to enhance
the differentiated characteristics of ß-catenin-induced ectopic
follicles, rather than to increase follicle number.
|
;
Pan et al., 2004),
demonstrating that crosstalk between the pathways is essential only in adult
epidermis.
We also observed contrasting effects of ß-catenin and Notch activity
in the IFE. ß-catenin activation does not alter proliferation or
differentiation of the IFE (Lo Celso et
al., 2004). By contrast, Notch activation resulted in a pronounced
increase in proliferation and in the total number of IFE layers. Although a
histologically normal cornified layer formed, keratin 10 expression was lost
in large patches of the IFE. The mechanism by which Notch signalling
influences the IFE remains to be explored, but it may involve signalling
between cells that differ in Notch ligand expression
(Lowell et al., 2000)
(Fig. 1G), or Notch activation
(Ross and Kadesch, 2004)
(Fig. 1B,C and
Fig. 6M-P).
Our studies place ß-catenin upstream of Notch in the epidermis and
show that Jag1 transcription is regulated by ß-catenin
signalling. Based on our observations and the known sites of epidermal Wnt
expression and activity (DasGupta and
Fuchs, 1999; Niemann and Watt,
2002; Reddy et al.,
2001), we propose that in the hair follicle pre-cortex
ß-catenin/Lef1 activity induces Jag1 expression, which then activates
Notch in the pre-cortex and underlying dermal papilla cells
(O'Shaughnessy et al., 2004;
Rendl et al., 2005) to promote
differentiation of hair follicle lineages
(Yamamoto et al., 2003).
Although Notch is an epidermal tumour suppressor
(Nicolas et al., 2003),
deletion of Jag1 does not have a major effect on epidermal
proliferation (Fig. 5). Notch
therefore differs from hedgehog signalling, which is also downstream of
ß-catenin, because hedgehog acts primarily to promote proliferation in
adult and embryonic hair follicles (Oro
and Higgins, 2003;
Silva-Vargas et al., 2005).
Thus, the mechanism by which ß-catenin triggers de novo hair follicle
formation in adult epidermis involves hedgehog-mediated proliferation and
jagged 1-mediated cell fate specification.
Epidermal self-renewal and differentiation involve both positive and
negative regulatory networks (Fuchs et
al., 2004). In the case of the ß-catenin/Notch interaction,
two potential mechanisms for limiting signalling, to maintain normal follicle
growth and differentiation, have already been identified. These are the
inhibition of Wnt4 expression by the Notch1 target gene p21
(Devgan et al., 2005), and
negative regulation of ß-catenin through its association with the Notch C
terminus (Hayward et al.,
2005). It seems likely that activation of the two pathways is
governed by feedback mechanisms in the epidermis.
In conclusion, we are only just beginning to appreciate the complex
interplay between individual signalling pathways that control adult epidermal
stem cell renewal and lineage selection. The Wnt and Notch pathways not only
intersect with one another, but also with other signalling pathways, including
integrins, Eph receptors and Bmps (Hasson
et al., 2005; Kluppel and
Wrana, 2005). The effects of signal strength and duration, and of
synergy and antagonism between pathways, must also be considered. Wnt and
Notch pathways are part of a network of multiple intersecting signalling
pathways that control the epidermal stem cell compartment.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
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|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
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